Methods for large-scale production of polypeptides, such as Factor VII polypeptides, in eukaryote cells are known in the art, see, e.g., WO 02/29083, WO 02/29084 and WO 03/29442. Although many of the problems associated with large-scale production of polypeptides, such as Factor VII polypeptides, have been overcome, a few problems still remain unresolved.
Maintaining the pH value of the culture liquid within a fairly narrow optimal production “window”, e.g. within 0.5 pH unit or less as it is for many cell cultures, is a particular problem. Generation of CO2 and lactate in the culture liquid causes a pH decrease, and for most practical purposes, addition of strong bases, e.g. 1 M NaOH or 1M Na2CO3, is necessary in order to stabilize pH or to maintain pH within a predetermined range. Addition of strong base however causes problems with respect to a dramatic, localized pH increase which may lead to anomalous cell metabolism (high rate of glucose consumption, high rate of lactate formation), if not cell apoptosis, cf. Nienow et al., Cytotechnology 22: 87-94, 1996; Langheinrich and Nienow, Biotechnology and Bioengineering, 66 (3): 171-179, 1999; Osman et al., Biotechnology and Bioengineering, 75 (1): 63-73, 2001; and Osman et al., Biotechnology and Bioengineering, 79 (4): 398-407, 2002.
Thus, there is a need for improved methods for large-scale production of polypeptides from eukaryote cells, in particular methods where the need for addition of strong base is reduced or eliminated.
Mostafa and Gu, Biotechnol. Prog., 2003, 19, 45-51, disclose various strategies for removing dissolved CO2 from large scale fed-batch cultures, e.g using sparging with air.
Pattison et al., Biotechnol. Prog., 2000, 16, 768-774, disclose measurement and control of dissolved CO2 in mammalian cell culture processes using an in situ fiber optic chemical sensor. Dissolved CO2 is removed by nitrogen sparging of the culture.
Although these references mention strategies for controlling the level of dissolved CO2 in cell culture vessels by means of gas sparging none of them mention that the need for base addition can in this way be reduced or totally eliminated.